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Abstract: FR-PO512

Hyperosmolarity-Induced Cell Shrinkage in Renal Cells: What a Role for RhoB?

Session Information

Category: Fluid, Electrolytes, and Acid-Base Disorders

  • 1101 Fluid, Electrolyte, and Acid-Base Disorders: Basic

Authors

  • Centrone, Mariangela, Universita degli Studi di Bari Aldo Moro, Bari, Italy
  • D'Agostino, Mariagrazia, Universita degli Studi di Bari Aldo Moro, Bari, Italy
  • Ranieri, Marianna, Universita degli Studi di Bari Aldo Moro, Bari, Italy
  • Venneri, Maria, Universita degli Studi di Bari Aldo Moro, Bari, Italy
  • Angelini, Ines, Universita degli Studi di Bari Aldo Moro, Bari, Italy
  • Di Mise, Annarita, Universita degli Studi di Bari Aldo Moro, Bari, Italy
  • Simone, Laura, Fondazione IRCCS Casa Sollievo della Sofferenza, San Giovanni Rotondo, Italy
  • Pisani, Francesco, Universita degli Studi di Bari Aldo Moro, Bari, Italy
  • Valenti, Giovanna, Universita degli Studi di Bari Aldo Moro, Bari, Italy
  • Tamma, Grazia, Universita degli Studi di Bari Aldo Moro, Bari, Italy
Background

The small Rho GTP-binding proteins are important regulators of cell morphology, function, and apoptosis. Unlike other Rho proteins, RhoB can be subjected to either geranylgeranylation (RhoB-GG) or farnesylation (RhoB-F), making that the only target of the farnesyltransferase inhibitors (FTIs). This study evaluated the involvement of RhoB farnesylation in the mechanism that regulates cell volume changes.

Methods

Mouse collecting duct MCD4 cells and human proximal tubule HK-2 cells were used as experimental models. Fluorescence Resonance Energy Transfer (FRET) studies were applied to evaluate the activity of RhoB. Cell volume was detected using an automated cell counter (Luna, Logos Biosystem), a calcein-based assay, and laser-scan confocal microscopy followed by 3D reconstruction and cell-volume analysis (3D-LSCM).

Results

Treatment with FTI-277 significantly reduced the cell volume in MCD4 and HK-2 cells. Compared to untreated cells, FTI-277 treatment altered hyperosmotic-dependent cell shrinkage response. FRET experiments revealed that RhoB was activated by hyperosmolarity regardless of FTI exposure. By contrast, hyposmolarity did not alter the activity of RhoB. To evaluate whether RhoB plays a role in volume reduction, cells were transiently transfected with RhoB-wt-EGFP and RhoB-CLLL-EGFP which cannot undergo farnesylation. Notably, hyperosmolarity caused a significant reduction in cell volume in mock and RhoB-wt-EGFP-expressing cells. By contrast, cells treated with FTI-277 or expressing the RhoB-CLLL-EGFP mutant did not properly respond to hyperosmolarity compared to mock and RhoB-wt-EGFP expressing cells. These findings were further confirmed by 3D-LSCM. Additionally, RhoB-CLLL-EGFP expressing cells showed relevant staining for Annexin-V compared to cells expressing EGFP and RhoB-wt-EGFP.

Conclusion

Together, these data suggest that: i) RhoB is sensitive to hyperosmolarity but not to hyposmolarity; ii) inhibition of RhoB farnesylation associates with an increase in cell apoptosis, likely suggesting that RhoB might be a paramount player controlling apoptosis by interfering with responses to cell volume change.

Funding

  • Government Support – Non-U.S.